Fig. 4: Fungal soluble β-glucan interaction with CR3 induces MEVsCa release.
From: Immune modulation by complement receptor 3-dependent human monocyte TGF-β1-transporting vesicles
a TGF-β1 is significantly upregulated in MEVsCa compared with MEVs, but not when CR3 was blocked (data are presented as mean values + /− SD, p = 0.0019, unpaired two-tailed t test, n = 3 donors). MEVs were isolated from 1-h-infected monocytes (5 × 105), and TGF-β1 was detected by ELISA. b CD11b knockout (KO) THP-1 cells generated via CRISPR-CAS9 lacks CD11b expression by both western blot and flow cytometry (see uncropped WB and gating strategy in Supplementary Fig. 7a, c). c TGF-β1 significantly increases in wild-type THP-1-derived TVsCa, but not EVs from CD11b KO THP-1 cells. EVs were isolated from uninfected or C. albicans-infected (1 h) wild-type or CD11b KO THP-1 cells (TVs or TVsCa, respectively) (each 5 × 105) (data are presented as mean values + /− SD, p = 0.0064, unpaired two-tailed t test, p = 0.0014, one-way ANOVA, n = 3 donors). d TGF-β1 significantly increases in ex vivo blood cell infection-derived vesicles (BCEVsCa) compared with control blood cell-derived vesicles (BCEVs) in wild-type mice (C57BL/6), but not CR3-deficient (CD11b KO) mice (B6.129S4-Itgamtm1Myd/J). In all cases, vesicles were isolated from 1-h-infected blood cells (1 × 107) (data are presented as mean values + /− SD, p = 0.0293, unpaired two-tailed t test, p = 0.0110, one-way ANOVA, n = 3 donors). e MEVsCa from wild-type but not from CR3-deficient mice show increased TGF-β1 concentration compared to MEVs (data are presented as mean values + /− SD, p = 0.0082, unpaired two-tailed t test, p = 0.0021, one-way ANOVA, n = 5 wild-type mice and n = 3 or CR3-deficient mice). Monocytes were generated from mouse bone marrow-derived stem cells. MEVs and MEVsCa (1 h infection) were each isolated from 5 × 105 monocytes. f CR3 (CD11b/ CD18) harbors two binding domains: the I-domain (binds iC3b) and the lectin-like site (LLS) domain (binds soluble β-glucan (sβG)) (modified from O’Brien et al.18). g MEVsSc-sβG and MEVsCa-sβG (induced for 1 h by sβG from S. cerevisiae and C. albicans, respectively) but not by iC3b show increased TGF-β1 concentrations compared with MEVs. EVs were isolated from activated monocytes (5 × 105) (data are presented as mean values + /− SD, p = 0.0053, p = 0.0227, p = 0.0077, unpaired two-tailed t test, n = 3 donors). h MEVs or MEVsCa-sβG tracking by DLSM. Data are representative of four independent experiments. i MEVsCa-sβG significantly increase compared with MEVs (data are presented as mean values + /− SD, p = 0.0023, unpaired two-tailed t test, n = 5 donors). j Size distributions of EVs determined with NanoSight NTA 3.2 software. EVs were isolated from 5 × 105 induced or control monocytes. Graphs were generated by overlaying the size distribution of MEVs and MEVsCa-sβG derived from five donors. k sβG from C. albicans binds to CR3 on monocytes as seen by proximity ligation assay (PLA). PLA staining– red: sβG /CD11b complexes, blue: DNA. Bars: 10 µm. Representative data of n = 5 experiments (five donors).
